Mapping of a palmitoylatable band 3-binding domain of human erythrocyte membrane protein 4.2

Abstract

Evidence accumulated over the years suggests that human erythrocyte membrane protein 4.2 is one of the proteins involved in strengthening the cytoskeleton-membrane interactions in the red blood cell. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemia. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3 (CDB3). In order to carry out an initial characterization of its interaction with the CDB3, protein 4. 2 was subjected to proteolytic cleavage and gel renaturation assay, and the 23-kDa N-terminal domain was found to interact with band 3. This domain contained two putative palmitoylatable cysteine residues, of which cysteine 203 was identified as the palmitoylatable cysteine. Recombinant glutathione S-transferase-fusion peptides derived from this domain were characterized with respect to their ability to interact with the CDB3. Whereas these studies do not rule out the involvement of other subsites on protein 4.2 in interaction with the CDB3, the evidence suggests that the region encompassing amino acid residues 187-211 is one of the domains critical for the protein 4.2-CDB3 interaction. This is also the first demonstration that palmitoylation serves as a positive modulator of this interaction.

Flow cytometric analysis of the association between blood group‐related proteins and the detergent‐insoluble material of K562 cells and erythroid precursors

“…Some interactions between cell surface and skeletal proteins of RBCs have been characterized in detail. For instance, the N‐terminal domain of Band 3 associates with ankyrin, protein 4·1 and protein 4·2, and the interacting domains of each protein have been mapped to some extent (Tanner, 1993; Lux & Palek, 1995 for reviews; Rybicki et al , 1995; Bhattacharyya et al , 1999). It has also been shown that the cytoplasmic tail of GPC, protein 4·1 and protein p55 form a ternary complex that is critical for membrane stability and the protein domains that interact have been precisely delineated (Nunomura et al , 2000).…”

Protein 4.2 interaction with hereditary spherocytosis mutants of the cytoplasmic domain of human anion exchanger 1

“…Protein 4.2 is a member of the TG (transglutaminase) family of enzymes, but has no catalytic activity [12]. It is myristoylated at Gly 2 [13] and palmitoylated at Cys 173 [14], which may allow association with cellular membranes. Protein 4.2 has been shown to associate with the plasma membrane of Sf9 insect cells [15] and Xenopus oocytes [16] in the absence of AE1.…”

“…In Figure 1 (right-hand panel), a homology model we constructed of protein 4.2 using human TG2 [17] as a template, is placed next to the crystal structure of cdAE1 to show their relative sizes. The N-terminal region of protein 4.2, encompassing residues 1−238, has been shown to represent the cdAE1-binding domain [14]. Residues 157−181 were found to be critical for the interaction and predicted formation of a β-hairpin in the centre of protein 4.2 with Cys 173 located at the bend.…”

Mapping of a spectrin-binding domain of human erythrocyte membrane protein 4.2

“…Band 3-protein 4.2-ankyrinspectrin interactions probably stablilize linkages between the cytoskeleton and the overlying membrane. Protein 4.2 associates in itro with band 3 [15,16], ankyrin [16] and spectrin [17]. Human erythrocyte protein 4.2 is N-terminally myristoylated [18] and palmitoylated [19], and its interaction with band 3 is modulated by palmitoylation [15].…”

“…Protein 4.2 associates in itro with band 3 [15,16], ankyrin [16] and spectrin [17]. Human erythrocyte protein 4.2 is N-terminally myristoylated [18] and palmitoylated [19], and its interaction with band 3 is modulated by palmitoylation [15]. It can be phosphorylated by a red-cell-membrane kinase that partially co-purifies with it, and has properties similar to the catalytic subunit of cAMP-dependent kinase [20].…”